A Faster Triphosphorylation Ribozyme

Abstract

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

Lower temperature optimum of a smaller, fragmented triphosphorylation ribozyme

“…We did not expect that the complete removal of the P5 stem would lead to functional ribozymes. 35 However, the fragmented ribozyme variants with truncated P5 duplex were fully functional, and two of them -with a P5 stem length of 2 and 3 base pairswere even faster than the ribozyme with a full P5 duplex (Fig. 3B).…”

“…This reduction suggested that the P5 helix fulfills a function during the triphosphorylation reaction, presumably by favoring the catalytically active conformation. On the other hand, the triphosphorylation rate of 0.15 min À1 for the construct without P5 helix (as compared to 0.31 min À1 for the parental construct 35 ) showed that structural elements can not only be reduced in length but also be completely omitted while maintaining a functional ribozyme.…”

“…These observations are also consistent with the finding that ribozyme variants with a base mismatch at the second position of a full P5 helix accelerate reaction kinetics. 35 Future studies with many different ribozymes may be able to determine whether this benefit of flexibility is specific for the studied ribozyme or whether it may be a general advantage of fragmented ribozymes. A general benefit for fragmented ribozymes would increase the likelihood of an RNA world emerging from a prebiotic scenario with short RNAs.…”

“…Triphosphorylation reactions were performed essentially as described. 34,35 Triphosphorylation reactions with 5.5 mM triphosphorylation ribozyme and substoichiometric concentrations of radiolabeled substrate were incubated with 50 mM Tmp, 100 mM MgCl 2 (corresponding to 50 mM free Mg 2+ ), and 50 mM Tris/HCl (final pH 8.1) for three hours at 22 1C if no other temperature is given. At specific time points, 1.5 mL of the reactions were added to 4 mL of formamide PAGE loading buffer containing 50 mM Na 2 EDTA, heat denatured, and separated by denaturing 20% PAGE.…”

Computational analysis of fitness landscapes and evolutionary networks from in vitro evolution experiments

“…For example, the Spinach2 aptamer [5] regulates the fluorescence of a fluorophore via binding, providing an interesting alternative to GFP tagging. In vitro selection has been applied to discover a variety of ribozymes [6], deoxyribozymes [7], aptazymes [8], and riboswitches [9]. With appropriate polymerase enzymes, selection experiments can even be done with non-natural nucleic acids, as shown for a series of xeno-nucleic acids (XNAs).…”